Project Summary The sudden emergence of Zika Virus (ZIKV) and the unexpected and serious complications associated with ZIKV brings into sharp focus how little we understand about the basic molecular virology of this virus. Although we can glean information from what we know of other mosquito-borne flaviviruses, it is clear that ZIKV has unique characteristics. One characteristic of the mosquito-borne flaviviruses is that during infection they produce a set of non-coding RNAs called subgenomic flaviviral RNAs (sfRNAs), which are 200-500 nucleotide long RNAs that are made of fragments of the 3? untranslated region (UTR) of the viral genomic RNA. These sfRNAs are the result of incomplete degradation of the viral RNA genome by a mechanism in which the virus elegantly ?co-opts? a powerful cellular exonuclease. Briefly, exonuclease Xrn1 loads on a subset of viral genomic RNAs and degrades them in a 5??3? direction until it encounters specific RNA sequences in the viral genome?s 3?UTR where the enzyme halts at precisely defined sites. The resultant sfRNAs inhibit innate immune pathways, bind RNAi-associated proteins, alter mRNA degradation patterns, and ultimately are direct drivers of pathogenesis. They may also be important in host adaptation as the virus moves between its invertebrate vector and vertebrate hosts. Currently, we do not know how many sfRNA species are produced by ZIKV, their sizes, amounts, or how this pattern changes in different host cells. This severely limits our ability to understand the mechanisms of ZIKV infection- associated pathogenesis, design experiments to explore sfRNA effects, or create strategies to alter sfRNA production. Furthermore, sfRNA?s disease-associated biological outcomes depend on the structure of the sfRNAs, but we have no map of the structure of the ZIKV 3? UTR or the sfRNAs that are derived from it. We propose exploratory research regarding the molecular basis of ZIKV infection and pathogenesis, focused on the sfRNAs. We propose two aims: (1) Determine the identity and pattern of ZIKV sfRNAs formed in cell types from different hosts, and (2) Determine the structural architecture of the ZIKV sfRNAs and how RNA structure drives sfRNA production. The proposed studies will provide fundamental knowledge regarding ZIKV sfRNAs. We will learn how to inhibit sfRNA formation (and thus make attenuated virus) through targeted mutation, providing knowledge and tools necessary for future research endeavors and useful for vaccine development. Our studies will also provide framework to interpret results from future studies, will establish a link between processes that occur in ZIKV infection to other FVs, and will create new knowledge applicable to future potential FV threats.